The availability of complete bacterial genome sequences and the elucidation of metabolic pathways have resulted in the use of such knowledge to engineer microorganisms for the production of compounds of industrial interest. Microbial production of industrial compounds requires the ability to efficiently engineer changes to the genomes of the organisms. Engineering changes such as adding, removing, or modifying genetic elements have often proven to be challenging and time consuming exercises. One such modification is genetically engineering modulations to the expression of relevant genes in a metabolic pathway.
There are a variety of ways to modulate gene expression. Microbial metabolic engineering generally involves the use of multi-copy vectors to express a gene of interest under the control of a strong or conditional promoter. This method of metabolic engineering for industrial use has several drawbacks. It is sometimes difficult to maintain the vector due to segregational instability. Deleterious effects on cell viability and growth are often observed due to the vector burden. It is also difficult to control the optimal expression level of desired genes on a vector. To avoid the undesirable effects of using a multi-copy vector, a general approach using homologous recombination via a single insertion of bacteriophage λ, transposes, or other suitable vectors containing the gene of interest has been used. However, this method also has drawbacks such as the need for multiple cloning steps in order to get the gene of interest into a suitable vector prior to recombination. Another drawback is the instability associated with the inserted genes, which can be lost due to excision. Lastly, these methods have a limitation associated with multiple insertions and the inability to control the location of the insertion site on a chromosome.
Although previous methods have been developed for making multiple DNA modifications in the chromosome, these have used transposes that are randomly integrated and require multiple cloning steps to insert genes of interest (Perdelchuk, M. Y., and Bennett, G. N. 1997. Gene. 187:231-238), or vectors that also require multiple cloning steps (PCT WO01/18222) and have not been applicable to all types of chromosomal modifications including insertions of whole genes or promoter sequences, deletions, and integrated transposes. Further, these methods have utilized a systematic approach to making multiple alterations at undefined loci as opposed to a combinatorial approach to making directed modifications on the chromosome.
The problem to be solved, therefore, is to define methods and materials to easily combine chromosomal modifications, created by any number of methods for chromosomal engineering, in one strain in a fashion that facilitates reaching optimum levels of product formation in bacteria, such as E. coli. The present invention has solved this problem by providing a method using P1 transduction and site-specific recombinase mediated marker excision to combine, in a linear, step-wise, and parallel combinatorial fashion chromosomal alterations. The present method allows for easy and efficient in vivo chromosomal engineering associated with biosynthetic pathway optimization.